Capsicum (Solanaceae or nightshade family) is a genus of plants, of which the sweet and aromatic fruits are used as a spice, a vegetable, and a medicine. The genus comprises about 40 species. Most varieties contain capsaicin (methyl vanillyl nonenamide), a pungent chemical that produces a strong burning sensation in the mouth and that can be used as a circulatory stimulant and pain reliever in medicine. The plants originate in Central and South America, but since they tolerate nearly every climate, the fruits are produced all over the world. Commercial peppers are primarily of the species Capsicum annuum (bell pepper, cayenne pepper, Jalapeño pepper, Anaheim pepper), Capsicum frutescens (Tabasco pepper) and Capsicum chinense (Habañero pepper).
Capsicum annuum, also known as paprika or pimento, is an herbaceous annual species with fruits that vary in length, color and pungency, depending upon the cultivar. The species is cultivated world-wide, for example in Western Europe and the United States of America (USA). Capsicum frutescens and C. chinense have small extremely pungent fruit and are used in tabascos and other hot pepper products. Since a fairly warm climate is necessary for a strong aroma, the species are cultivated primarily in tropical regions and in warmer regions of the USA.
Vegetable crops of Capsicum spp. are damaged by many Tobamoviruses during cultivation. The genus Tobamovirus includes the type species Tobacco mosaic virus (TMV), and the serologically related Tomato mosaic virus (ToMV), Pepper mild mottle virus (PMMoV) and several other plant viruses. The viral infections can reduce the plant's vigor but do not commonly kill it. TMV, for instance, which affects a wide range of plants, including pepper, tomato and eggplant, causes severe necrosis on pepper fruits leaving most of the fruits unmarketable. TMV is a highly persistent disease because it can remain viable in soil for many years.
PMMoV systemically infects all Capsicum spp., including cultivars that are resistant to TMV and ToMV. Symptoms of the disease in bell pepper plants include stunting of young plants, crinkling and yellow mottling of leaves. Fruits are malformed (lumpy and mottled) and slightly reduced in size.
In 1980 a viral strain of a TMV-like virus (tm-3) was isolated that could infect the PMMoV-resistant Capsicum lines known at that time (Boukema et al., 1980). The isolated strain was called strain P14 of TMV. Following the discovery of strain P14, a line of the plant species C. chacoense (line PI 260429) was found in 1982 that exhibited resistance to this new virus (Boukema, 1982). It was later determined that the resistance in this plant was conferred by the L4 allele of the L-locus (Boukema, 1984) and that strain P14 was able to overcome the former resistance conferred by the L3 allele, whereas it could not break the resistance conferred by the L4 allele. This L3-conferred resistance-breaking strain, originally classified as TMV, was later reclassified as a specific PMMoV pathotype and the pathotype was assigned pathotype 1.2.3.
Amongst breeders, the C. chacoense line that exhibits resistance to PMMoV pathotype 1.2.3 as well as commercial pepper lines derived thereof, are commonly referred to as Tm3-resistant lines. Many breeding companies have since introgressed genetic material of this C. chacoense line comprising the L4 allele into their breeding lines in order to obtain resistant pepper plants with commercially favourable characteristics.
It is now known that the L4 allele confers resistance to a number of viruses, including ToMV, TMV, and to the PMMoV pathotypes 1, 1.2, and 1.2.3. The resistance conferred by the L4 allele is hereinafter termed “PMMoV resistance”, although strictly speaking it confers broader resistance, as described above.
The nomenclature of Tobamoviruses has been the subject to several revisions in the period between 1980 and 2004. Herein, use is made of the nomenclature of the “Guidelines for the conduct of Tests for Distinctness, Uniformity and Stability” (TG/76/7) for sweet Pepper (C. annuum L.) issued by the Union for the Protection of New Varieties of Plants (UPOV) on Apr. 11, 1994. In these guidelines the genetic resistance to pepper Tobamovirus pathotypes is considered to be controlled by 5 alleles (L−, L1, L2, L3, and L4) located on the same locus (L-locus). Reference is herein explicitly made to the Table on pp. 21-22 of the above-referred UPOV guideline TG/76/7, wherein the relationship is shown between resistance against the various virus pathotypes and the resistance-conferring allelic compositions in pepper. A homozygous L3L3 genotype of C. annuum confers resistance to Tobacco Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV), Bell Pepper Mosaic Virus (BePMV), Tobacco Mild Green Mosaic Virus (TMGMV), Dulcamara Yellow Fleck Virus (DYFV), and Pepper Mild Mottle Virus (PMMoV) pathotype 1.2, whereas the homozygous L4L4 genotype provides an additional resistance to Pepper Mild Mottle Virus (PMMoV) pathotype 1.2.3.
The UPOV nomenclature differs somewhat from the nomenclature usually employed in the scientific literature on virology. Generally in the scientific literature nomenclature is confined to the species name and the designation of the isolate. The pathotype designation is only very occasionally described. For instance, the pathotype 1.2.3 of PMMoV is not incorporated in The Universal Virus Database of the International Committee on Taxonomy of Viruses (ICTV). It should further be noted that with respect to nomenclature and the developments therein over time, a given Tobamovirus may have acquired two or more trivial names and may have been further reclassified as explained in more detail below. It is to be understood that irrespective of any new assignment of a viral isolate, the present invention relates to resistance of plants to any viral strain that is conferred by the L4 allele.
Is indicated above, the resistance of Capsicum spp. to Tobamoviruses is conferred by pathotype-specific alleles (L1, L2, L3, L4) of the L-gene, and resistance to PMMoV pathotype 1.2.3 is conferred by the L4 allele. The region where the L-locus is located is positioned on the telomere of chromosome 11 south (Lefebvre et al, 2002). The resistance alleles act via triggering of the hypersensitive response (HR).
A remarkable and undesired characteristic of the resistance conferred by the L4 allele when introgressed from C. chacoense into other Capsicum species, is that the resistance in the new plant lines is inherited non-Mendelian. Mendel's law of segregation states that allele pairs segregate during gamete formation, and randomly unite at fertilization. For each character, a diploid organism inherits two alleles, one from each parent. If the two alleles differ, then one, the dominant allele, is fully expressed in the organism's phenotype; the other, recessive allele has no noticeable effect on the phenotype. According to normal Mendelian inheritance, the cross of a plant with a homozygous dominant allele and a plant with a homozygous recessive allele will result in an F1, or first filial population, that is uniform, both genetically (whole population is heterozygous) as well as phenotypically (whole population expresses dominant trait). Such an F1 population is said to be non-segregating for that dominant trait (of course in the F2 population segregation will occur). Thus, an F1 population does not segregate for a dominant trait when at least one of the parents is homozygous for that trait. Likewise, the presence of a non-segregating F1 confirms the homozygosity of a dominant trait in one of a parent lines. This is however not the case with commercial plant lines in which the L4 allele is introgressed.
For plant breeders it is important that breeding lines are homozygous (true breeding), because the result of the breeding should preferably be predictable. When, for instance, a commercial hybrid is produced from two (homozygous) inbred lines, the resulting heterozygous F1 plants are fitter than their inbred parents as a result of hybrid vigor or heterosis. Plant breeders purposely exploit such heterotic crosses to generate more robust progeny and the homozygous inbred lines serve as producer lines with high economic value. In breeding practice therefore, it is customary to assess whether a plant line is homozygous for a dominant resistance trait conferred by a single gene (i.e., a monogenic dominant trait) by selfing that plant line and screening its offspring for non-segregation. Alternatively an expected homozygous dominant plant line can be crossed with a homozygous recessive line whereby a segregating F1 reveals that the tested “dominant” parent is not homozygous.
The L4 resistance allele is believed to be a normal monogenic dominant resistance allele (Boukema, 1983; Van Duin, 1998). However, in the case of the PMMoV-resistance conferred by the L4 resistance allele, breeders generally observe problems with the predictability of crosses from homozygous lines and the stability of those lines. Whereas the selfing of an expected homozygous (L4L4) resistant plant invariably results in an offspring with a uniform resistant phenotype (thereby confirming the homozygosity of the parent plant), the crossing, on the other hand, of such a homozygous resistant parent plant with a susceptible parent plant (i.e. one that lacks the L4 allele) often results in an F1 comprising both resistant and susceptible plants, i.e. in a segregating F1. The susceptibility of the plants may for instance be detected by the presence of systemic mosaic after inoculation with PMMoV pathotype 1.2.3. This phenomenon, wherein resistance is surprisingly lost in some of the F1 offspring plants, is very inconvenient to breeders for reasons outline above. Breeders often refer to such unpredictable homozygous parent plants as “segregating” plants, although strictly speaking, their F1 offspring is segregating.
In the development of improved PMMoV-resistant inbred lines, and by using conventional plant selection and test crossings, breeders have recently shown to be able to obtain homozygous plants that produce a normal non-segregating F1, (i.e., one that follows Mendel's law), thereby having fixed the resistance conferred by the L4 allele in that line. It is at present unclear how this was achieved. A major problem of these “non-segregating” plants is, however, that they exhibit reduced fertility and dwarf growth. An important consequence of the reduced fertility is that such plants produce limited amounts of seed and/or limited amount of pollen. Thus, although such plants are resistant and “non-segregating”, they exhibit a phenotype characterized by a low fertility and dwarf growth that is undesired for commercial seed production. The dwarf growth may for instance be indicated by late ripening of the seed (i.e. slow growth) or by reduced (rate of) development of the plant. This phenotype will be further designated in this application as an “SNFD phenotype”, for Segregating Non-Fertile Dwarf. There is at present no explanation for the association between these negative traits, which include problems with growth, fertility and sterility, and the introgression of the L4 allele from C. chacoense into other Capsicum lines (vide, e.g. Van Duin, 1998).
The introgression of any of the L1, L2 and L3 alleles of the L-gene into Capsicum spp. have always been found to produce Mendelian inherited populations without showing the SNFD phenotype in homozygous resistant plants. The L4 introgression from C. chacoense now for the first time gives rise to the above problems.
At present there is a need for pepper plants that are resistant to PMMoV pathotype 1.2.3 as conferred by the L4 allele, that are “non-segregating”, that show normal (rates of) development and that produce proper amounts of seed. It is therefore an object of the present invention to provide plants of the genus Capsicum, which combine resistance to PMMoV pathotype 1.2.3 as conferred by the L4 allele with agronomically or commercially good traits, such as good growth and normal seed production, thus without the undesired characteristics of the SNFD phenotype.